z Report on the AOAC ERP for Sugars and Fructans AOAC First Action Method z Fructan (Inulin, FOS, Levan, and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients) z Method Scope

§ [This method is based on AOAC Method 999.03, which is commercially available from Megazyme as the Fructan Assay Kit (Megazyme Cat. No. K-FRUC).] See Table 2018.07 for method performance study supporting acceptance of the method. z Method Principle

Using this method, these forms of fructans are extracted from pet foods, feeds, and ingredients into boiling water. Sucrose, starches, and maltodextrin are concurrently enzymatically hydrolyzed to glucose and fructose. All reducing sugars are then converted to sugar alcohols, thus removing them as potential interferences. The method states native fructans and non-reducing FOS are not affected by these reactions. The SMPR- listed fructans are hydrolyzed to fructose and glucose using a mixture of three enzymes to cover the different possible fructan internal sugar linkages. Quantitation of the total amount of reducing sugars (fructan- derived fructose and glucose) is performed using the PAHBAH reducing sugar method (PAHBAH = p-hydroxybenzoic acid hydrazide). A single point calibration of absorbance equivalent to 54.5 µg fructose is used.

First Action AOAC Method z Sugar Profile by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection Validation package was extensive - 130 pages z Has not yet been published in the OMA. Should be available soon. z Method Scope

§ The validation study established that the method is fit for the purpose of analyzing nutritional sugar compounds in a broad range of human food, dietary supplements, and pet/animal feeds covering all sectors of the AOAC compositional triangle, including different moisture content and carbohydrate sources. z Method Principle

§ Using this method, sugars are extracted in 50:50 water: ethanol and optionally further clarified with Carrez. Extracts are then filtered, dried, and reconstituted in water. The test solution is analyzed by HPAEC-PAD (high pressure anion-exchange chromatography with pulsed amperometric detection) equipped with post column reagent delivery pump against a 5-point linear internal standard corrected curve.

Summary of matrix categories, matrices, and samples used in the multi-lab validation study

z AOAC food Matrix Category Matrix Material ID Sample Type Validation site pyramid sector

Fat/Oil Palm oil 1 Innate, spikes MSN Baking chocolate 2 Innate, spikes MSN High fat Peanut butter 3 Innate, spikes SGP, HAR Lunch meat Salami 4 Innate, spikes BC Cereal NIST 3233 5 CRM BC, HAR, MSN, SGP Lemon Juice 5 Innate, spikes BC, HAR, MSN, SGP Food Products Artificial Foodstuff BCR-644 5 CRM BC, HAR, SGP Vegetable Spinach 7 Innate, spikes MSN Lunch meat Turkey 7 Innate, spikes BC Lunch meat Ham 8 Innate, spikes BC Meat Mince 9 Innate, spikes HAR Seafood Tuna 9 Innate, spikes BC Infant Formula and Adult Nutritional Infant formula NIST 1849a 6 CRM HAR, MSN, SGP Gummy 5 Innate, spikes MSN Tablet 5 Innate, spikes MSN, HAR Dietary Supplements Premix 5 Innate, spikes MSN Protein powder drink 6 Innate, spikes MSN Horse feed 5 Innate, spikes MSN Swine feed 5 Innate, spikes MSN Milk replacement supplement 6 Innate, spikes MSN Pet Food and Animal Feed Dry dog food 6 Innate, spikes MSN Dry cat food 7 Innate, spikes MSN, HAR Wet cat food 7 Innate, spikes BC Validation site codes: BC = Battle Creek, MI, USA; MSN = Madison, WI, USA; HAR = Harrogate, UK, SGP = Singapore z Summary of CRM/SRM accuracy validation data

Individual Lab RM Range Lab Overall Matrix Sample ID # of labs Reps Analyte RM Range (%) Average Range (%) Mean (%) (%)

Fructose 0.81 0.42 - 1.20 0.727 0.694 -0.764

Glucose 1.04 0.68 - 1.40 0.923 0.906 -0.938

Cereal NIST 3233 4 45 Sucrose 13.42 12.67 -14.17 13.6 13.2 - 13.9 0.415 -0.485 Maltose 0.46 0.37 - 0.55 0.443

Total Sugar 15.8 14.3 - 17.3 15.7 15.2 - 16.0

Fructose 16.2 15.1 - 17.3 15.8 15.6 - 16.0

Artificial Foodstuff BCR-644 3 27 Sucrose 10.81 10.56 -11.06 10.7 10.5 – 10.8 Lactose 15.85 15.56 -16.14 15.6 15.4 – 15.8 Monohydrate Lactose Infant formula/Drink NIST 1849a 3 27 47.6 42.1 - 53.1 49.5 48.0 – 50.7 Monohydrate Bold indicates individual result outside established range. z Summary of intermediate precision (%RSDi) and reproducibility (%RSDR) validation data

%RSDi range # of Total # or Matrix Material ID Analyte (individual lab %RSDR labs replicates precision) Fructose 0.9 – 7.3 6.6 Glucose 1.3 – 3.2 3.3 Cereal NIST 3233 4 45 Sucrose 2.0 – 4.1 3.5 Maltose 1.5 - 7.1 7.6 Total Sugar 1.7 – 4.0 3.4 Fructose 1.1 – 2.9 2.5 Artificial Foodstuff BCR- 3 30 Sucrose 1.9 – 2.9 2.6 644 Lactose Monohydrate 1.9 – 3.3 2.8 Infant formula NIST 1849a 3 27 Lactose Monohydrate 1.8 – 6.0 4.5 Glucose 1.2 – 3.2 2.7 Lemon juice 4 36 Fructose 0.9 – 4.0 3.3 Total Sugar 1.8 – 2.4 2.2 Peanut butter 2 18 Sucrose 2.5 – 5.1 3.9 Sucrose 2.5 – 2.6 2.6 Tablet 2 18 Maltose 3.0 – 6.7 5 Total Sugar 2.5 (both labs) 2.6 Summary of intermediate precision (%RSDi) validation data

z Total # or 9 Glucose 4 9 Lactose 1.5 Matrix # of labs replicate Analyte %RSDi Protein powder drink 1 s 8 Maltose 7.2 Total Sugar 1.7 Glucose 2.9 9 Spinach 1 9 Sucrose 4.4 Glucose 1.0 Sucrose 1.5 Total Sugar 2.8 Dry Dog Food 1 9 Fructose 1.2 Baking chocolate 1 9 Sucrose 2.5 Total Sugar 1.7 Glucose 2.3 Dry cat food 1 9 Sucrose 1.6 Ham 1 9 Sucrose 1.7 Glucose 3.0 Total Sugar 0.9 Wet Cat Food 1 9 Maltose 2.9 Glucose 4.5 Total Sugar 3.0 Salami 1 9 Sucrose 2.4 Fructose Total Sugar 1.9 1.7 Glucose 1.1 Fructose 1.9 Horse Feed 1 9 Sucrose 7.0 Glucose 3.5 Maltose 4.8 Turkey 1 9 Sucrose 1.1 Total Sugar 1.6 Maltose 2.5 Fructose 3.0 Total Sugar 0.8 Glucose 2.0 Mince 1 9 Glucose 2.9 Sucrose 3.4 Swine Feed 1 9 Fructose 2.6 Lactose 2.3 Glucose 1.3 Maltose 3.1 Gummy 1 9 Sucrose 1.3 Total Sugar 2.2 Maltose 1.2 Galactose 0.7 Total Sugar 1.3 Glucose 1.9 Milk Replacement Glucose 2.5 1 9 Lacotse 5.8 Supplement Sucrose 2.2 Maltose 5.6 Premix 1 8 Maltose 1.7 Total Sugar 5.7 Total Sugar 2.4 Bold indicates individual result outside AOAC 2018.001 SMPR z z Questions? z

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§NancyThiex and John Szpylka

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